The elucidation of the ubiquitin/proteasome system in eukaryotes has greatly contributed to our understanding of how cells work. However, many mechanistic features of how 26S proteasomes function remain unclear. One key question involves the function of the six AAA ATPases found in the 19S regulatory particle of the proteasome. Despite the importance of these proteins in proteasome function, very few studies have directly addressed the specific roles of ATPase activity in proteasome function. The goal of this proposal is to apply a novel 'chemical genetics' approach that will enable us to systematically deduce the molecular function of each ATPase in the 19S. Inhibitors are generated by chemically modifying purine derivatives with bulky substituents to complement a rationally designed enlargement of the ATP-binding sites of the target ATPases. While the 'analogue-sensitive' ATPases can bind to either normal ATP or the ATP analogue, wild-type ATPases are unaffected in the presence of ATP analogue, allowing specific inhibition of a given ATPase in both in vitro and in vivo assays. Initially we will characterize the roles of the yeast 19S ATPases Rpt1 and Rpt5 in proteasome function, with the goal of eventually characterizing all six ATPases.